1. Field of the Invention
The present invention is directed to a new nucleic acid molecule that is codon-optimized to express beta interferon in Escherichia coli with greater efficacy.
2. Description of the Related Art
Many proteins or polypeptides are known that hold great promise for use in treating a wide variety of diseases or disorders. Interferons are relatively small polypeptide proteins which are secreted by most animal cells in response to exposure to a variety of inducers. Because of their antiviral, antiproliferative and immunomodulatory properties, interferons are of great interest as therapeutic agents. They exert their cellular activities by binding to specific membrane receptors on the cell surface. Once bound to the cell membrane, interferons initiate a complex sequence of intracellular events. In vitro studies demonstrated that these include the induction of certain enzymes, suppression of cell proliferation, immunomodulating activities such as enhancement of the phagocytic activity of macrophages and augmentation of the specific cytotoxicity of lymphocytes for target cells, and inhibition of virus replication in virus-infected cells. Thus, interferon proteins are functionally defined, and a wide variety of natural and synthetic or recombinant interferons are known. There are three major types of human interferons (“IFNs”). These are: Leukocyte IFN or IFN-alpha, a Type 1 IFN produced in vivo by leukocytes.
Fibroblast IFN or IFN-beta, a Type 1 IFN produced in vivo by fibroblasts.
Immune IFN or IFN-gamma, a Type 2 IFN produced in vivo by the immune system.
IFN-beta is of particular interest for the treatment of a number of diseases or disorders, and especially in the treatment of multiple sclerosis or MS. Natural human IFN-beta is a 166 amino acid glycoprotein, and the encoding gene has been sequenced by Taniguchi, et al. al., 1980, Gene 10: 11-15, and R. Derynck, et al., supra. Natural IFN-beta has three cysteine (cys) residues, located at amino acid positions 17, 31 and 141, respectively. In addition, numerous recombinant variants of IFN-beta are known.
Three recombinant IFN-beta products are licensed in Europe and the U.S. for treatment of MS. These are interferon beta-1a (“IFN-beta-1a ”) or Avonex® (Biogen, Inc., Cambridge, Mass.), another IFN-beta-1a product marketed as Rebif® (Ares-Serono, Norwood, Mass.) and Ser17 interferon-beta-1b (“IFN-beta-1b Ser17”) or Betaseron® (Berlex, Richmond, Calif.).
IFN beta-1a is produced in mammalian cells, e.g., Chinese Hamster Ovary (“CHO”) cells using the natural human gene sequence, and the produced protein is glycosylated. See, for example, U.S. Pat. Nos. 5,795,779, 5,376,567 and 4,966,843, incorporated by reference herein. IFN beta-1b Ser17 differs structurally from IFN-beta1a (Avonex® and Rebif®) because it is produced in Escherichia coli (“E. coli”) using a modified human gene sequence having an engineered cysteine-to-serine substitution at amino acid position 17, so that the protein is non-glycosylated. See, e.g., U.S. Pat. Nos. 4,588,585 and 4,737,462, the disclosures of which are incorporated by reference herein.
Both Rebif® and Avonex® are stated by their package inserts to have specific activities, by differing methods, of at least 2-3×108 international units (IU)/mg. The Betaseron® package insert reports a specific activity of approximately 3×107 IU/mg, indicating a ten-fold difference in potency. While these activities are determined by somewhat different methods, the order of magnitude differences in antiviral and antitumor activities are also reflected in the recommended doses, which are measured in micrograms (60-130 mcg/week) for the Rebif® and Avonex® glycosylated IFN-beta 1a products, and from 0.25 milligrams and up for the non-glycosylated Betaseron® IFN-beta 1b.
IFN-beta, in each of its recombinant formulations, has multiple effects on the immune system, including the ability to inhibit viral replication. IFN-beta-1b is described by the manufacturer (Berlex, Richmond, Calif.) as enhancing suppressor T cell activity, reducing proinflamatory cytokine production, down-regulation of antigen presentation, and inhibition of lymphocyte trafficking into the central nervous system. Other sources have reported that IFN-beta reduces the production of IFN-gamma by T-lymphocytes. Other beneficial therapeutic effects are also suspected.
However, production of recombinant proteins in cell culture remains an expensive process. For this reason, there remains a longstanding and heretofore unsolved need in the art for significantly improved vectors and methods of producing IFN-beta compositions, particularly those containing IFN-beta 1b, particularly in non-mammalian cells.